The order of the steps in the Start Run wizard has been adjusted to automatically set several parameters of the sequencing run depending on the sequencing kit used.

GeneReader Software now supports the use of 9-mer barcodes for panels using the unique molecular index (UMI) technology (read design V3) and 6-mer barcodes for non-UMI (read design V2) panels.

If the barcode of a flow cell is not readable while loading the flow cell into the GeneReader Instrument, the user can now either retry scanning the flow cell or enter a custom barcode manually.

If the Windows operating system requires a restart due to the installation of a security patch, GeneReader Software now shows this information right after closing the Run Finished or Unload wizard. This information message offers the options of performing the system restart before starting the next sequencing run.

The GeneReader Workstation Windows operating system has been updated to include the latest security patches.

This release of GeneRead Link 2.0.0 includes major changes in the architecture and user interface to support parallel workflows. Workflows are now part of an NGS 1.1 workflow extension plugin. The version of the extension is visible in the Configuration/Workflow Management section.

Main features for this release

User interface updated to support complex workflows with multiple paths

A new work-to-do view as entry page. This is now split into queued samples/in process/in approval, showing samples from all experiments across all workflows . The work-to-do view provides an overview of all samples processed in different areas of the workflow, while at the same time being able to filter the sample status at specific areas of the workflow.

Should the GeneRead QIAcube connection be lost, it is now possible to skip the rest of the GeneRead Link setup of the Clonal Amp experiment, so that the user can continue manually on the GeneRead QIAcube.

Character restriction and input validation on sample and pool IDs aligned with that of the GeneReader Instrument.

Support of the new UMI (V3)) frontend workflow.

QIAcube and manual Library Preparation supported for DNA and RNA based samples.

RNA Extraction and Reverse Transcription supported.

Assay/chemicals changes.

Updated/new panel configurations.

ATP renamed to AIT.

GeneRead QIAact Lung Panels are part of the distribution package.

Changes in Clonal Amplification experiment setup to be in alignment with new Library Preparation protocols – only normalized libraries are accepted as input for Clonal Amplification experiments.

The previous issue in which under certain conditions GeneRead Link stopped updating the QCI Analyze status, has now been fixed. As a result, all changes in QCI Analyze sample status are reflected by GeneRead Link.

In the previous version of GeneRead Link there was a bug causing too many samples to be failed in the Clonal Amplication step. In this workflow step samples are assigned to four library pools. In the step 'Perform Emulsion PCR' with run status set to 'Run passed without errors' for one plate and 'Run failed' for other plate (after the experiment is finished) all samples previously showed a PCR failed flag in the approval queue. This bug has now been fixed.

Pool names that were already used in one Clonal Amplification experiment are now no longer allowed to be used within an alternate experiment. Previously, in case the same pool was used in another Clonal Amplification experiment at the same time, GeneReader would show the pool name twice, but with different samples.

Corrected LIMS report HL7 messages for samples being released without results, now contain the name of the user that released the samples.

The system now prevents different gene panels from having the same short name. Previously, this was possible and lead to a system error when defining tests for these panels.

All analysis workflows have been optimized and re-configured for use with the GeneRead UMI Advanced Sequencing Q Kit. See tables 1 and 3 of the QCI Analyze 1.4.5 user manual for details on parameter settings.

AIT FFPE and plasma analysis workflows - updated Regions of interest and Variants of interest to include additional Regions and Variants of interest:

ERBB2 c.2310_2311insGCATACGTGATG

ALK c.3604G>A

KRAS c.33_34delTGinsCT

In the Variant of interest list, deletions and insertions are now left-aligned. This aligns with how QCI Analyze calls variants and ensures that detected indels are appropriately annotated as being variants of interest. This change does not impact variant calling, only annotation of called variants.

Lung DNA FFPE and plasma analysis workflows:

Enhanced detection of insertions and deletions in complex regions via improved realignment of the read mapping.

Option to disable individual analysis workflows if these are not used.

Option to delete single samples, available from Sample analysis details panel.

GeneReader Planner

The Adapter Q drop-down reflects the use of 9-mer barcodes (9mBC) for panels using the unique molecular index (UMI) technology and 6-mer barcodes (BC) for non-UMI panels.

Comparison tool

Selection of analysis results for a comparison is now done via an intuitive multi-select list.

Comparison reports have been divided into tabs for ease of view.

Deep-links for individual comparisons are now available allowing you to refer colleagues to a specific comparison.

The comparison tool is now available to all users, not just administrators.

Comparison reports can now be exported to Excel.

Comparison jobs now run instantly as opposed to being queued behind analysis workflows.

Sample analysis details panel

Analysis results now features CNV results.

Error message presented for failed sample analyses.

Download options included in panel - VCF, PDF, Excel, ZIP archive.

Link to QCI Interpret for GeneReader test is now displayed as a link inside panel (was previously displayed as a button below).

Upload button available for previously uploaded sample analysis.

Sample analysis report

Summary section:

Comments moved up to more prominent position.

Analysis pass/out-of-spec info removed.

‘Variant status’ renamed to ‘Analysis results’ and coloring removed.

Quality control section:

Sample index and UMI included in average read length.

Clear indication of whether coverage is based on raw reads or UMI.

Variants section:

F/R test given as scientific value if below 0.01.

Introduction of average base quality column (Avg Q).

History section:

Minor adjustments to log messages.

Improved enable/disable scroll functionality in track viewer.

Download options available via one 'Download' button.

PDF report: Green/yellow/red coloring of rows removed.

Bug fixes

Under rare circumstances, the variant Qual value could not be calculated and the resulting N/A were mistakenly represented in the variant table as the value zero. This issue has now been fixed: Now, if the Qual value cannot be calculated, the table column is left empty.

The Lung DNA analysis report now includes all genes, including those designated targets for CNV detection only, in the QC tables Detected variants, Read coverage and UMI read coverage. This ensures concordance between the sum of variants in the Detected variants table and the sum of variants in variant tables 3.1 and 3.2. Previously, a discrepancy between these could be observed if an analysis workflow was configured to 'Detect variants outside ROI'. This change does not impact the number of reported variants in the variant tables.

Previously, Variants of interest deletions that were tested and showed to not be present in the sample wrongfully were listed in table 3.3 as being 'Not tested'. This bug has now been fixed.

Known issues

Qual value missing for some variants

Description:
In rare cases the variant Qual value cannot be calculated due to an underlying issue. Such variants will appear in the QCI Analyze variant tables with an empty Qual field. The underlying NaN value is visible form the track viewer pop-up info box. We are working to address this in a future version.

For genes on the positive strand, variant coding impact for duplications of intron-exon splice sites may be wrong.

Description:Genomic duplications of the intron-exon splice site - an event that effectively adds bases to the exon codon region and thus changes the amino acid sequence - will be reported in QCI Analyze as an insertion. As insertions in QCI Analyze by default are left-aligned relative to the reference genome, for genes on the positive strand the duplication can in rare cases be attributed to the intro and consequently, the mutation will be reported incorrectly as having no amino acid impact. For genes on the negative strand, the duplication will be attributed to the exon, and the corresponding amino acid impact correctly assigned.

Based on the QIAGEN Knowledgebase, we could identify the following pathogenic variants in BRCA2 associated with hereditary breast cancer:

BRCA2 p.A2603Gfs*46

BRCA2 p.Asn2879Lysfs

Pathogenic variants in somatic cancer, which are affected by this issue, could not be identified for the current GeneReader QIAact panels and therefore the risk that this issue will have an impact for you is very small.

How to identify potentially affected variants:

1. In the variant table column 'Type', look for 'insertions'. Select the 'Filter' item to access the 'Advanced Filter' and filter for ‘Type/contains/Insertion’.
2. Look for variants with no p. variant annotation (indicating placement in non-coding area). Sort by clicking the column header.
3. In the column ‘c. variant’, look for annotations signifying placement in the 3’ end of an intron. The format will be ‘c.ex-in’, ‘ex’ and ‘in’ being digits (e.g. c.88-3). ‘ex’ indicates the first position in the downstream exon, ‘-‘ (minus) that the insertion is placed 5’ of this exon, and ‘in’ the position in the intron measured from the splice site. Use the Advanced Filter to filter for ‘c.variant/contains/-‘. If any variants remain, inspect the ‘in’ digit. Only variants for which ‘in’ is smaller than the length of the insertion will potentially be affected by this issue.
4. Check to see if the variant is located in a gene on the positive strand. Select the variant and inspect the blue gene annotation bar in the right-hand side track viewer. Zoom out to view the full gene. If located on the positive strand, the gene annotation bar will point to the right. If a variant is located in a negative strand gene it will not be affected by this issue.
For variants, which, based on the above, are flagged as potentially affected by this issue, check the read mapping to determine if the insertion is in fact a duplication covering the intron-exon splice site. If so, note that this variant will in fact give rise to amino acid changes and should be considered for downstream interpretation.